The mechanism of poliovirus RNA replication is being examined in this study. The poliovirus RNA dependent RNA polymerase has been isolated from the cytoplasm of infected cells as a soluble and template-dependent enzyme. The purified polymerase can synthesize a heteropolymeric and complementary copy of polio virion RNA in vitro. The product RNA is being characterized for size by electrophoresis on agarose gels containing CH3HgOH and for its sequence composition by two-dimensional "fingerprinting" of RNase Tl-generated oligoribonucleotides. The polypeptide composition of the purified polymerase is being examined. Only one viral protein with a molecular weight of 62,500 (p63) is required for elongation activity when oligo (U) is used as a primer. We plan to define other factors that may be required to initiate RNA synthesis in the absence Ff added oligo(U). In particular, we will investigate if the protein covalently linked to virion RNA (VPg) plays a role in initiating RNA synthesis. The relative efficiency of plus and minus strand poliovirus RNA to function as a template with the purified enzyme will also be examined. This may help to explain the asymmetric mechanism of viral RNA replication.